Our long term goal is to understand early vertebrate development at the molecular level. We study the problem in the frog Xenopus, whose abundant eggs are large and readily manipulated by microinjection and microsurgery. The eggs are large enough to produce material for biochemical analysis, and importantly for this project, ample RNA from manipulated embryos for deep sequencing. A combination of experimental embryology and molecular manipulation provides approaches to the roles of specific signaling pathways, transcriptional and post- transcriptional regulation in elaborating the structure of the embryo. Most of the paradigms for development of vertebrate embryos have come first from work with amphibians, and many of the signaling pathways were first analyzed using amphibian embryos. Gain of function experiments using mRNA injection, and loss of function using morpholino oligonucleotides have provided insights into the mechanisms that underlie tissue differentiation and morphogenesis. During previous grant periods, we have used expression cloning to identify potent signaling and signal transduction activities that contribute to embryoni development. In screens for embryonic activities that alter neural patterning we identified several RNA regulators that show specific effects in both gain of function and loss of function experiments. We have established methods to study both splicing regulation and changes in gene expression using deep sequencing. In aim 1 of the next grant period, we will continue analysis of selected RNA binding and pre-mRNA splice regulators that are deployed during the induction and patterning of the neural plate, and will continue to gain new insight into the selective effect of these proteins on specific splicing choices. In addition to splicing regulation we will also study transcription factors, and chromatin regulators whose expression are altered during neural patterning, and will particularly investigate factors that are turned on by the Wnt pathway during posterior patterning of the neural plate.